No TPM values, no reads processed

[marco91sol]

Hi,

I am using TPMCalculator using the following command:

TPMCalculator -g file.gtf -d ./ -a -p -e

In the current directory, I have sorted bam files deriving from HISAT2 alignment tool of RNA reads against the genome.

Here, you will find the head of samtools view on bam file.

Here, follows part of the log file:

Reading GTF file ... Done in 12.0684 seconds Parsing sample: trimmed_hisat_out.bam.sorted 0 reads processed in 23.4511 seconds Printing results Total time: 42.3443 seconds

Thanks for the support!

Br, Marco

[pongorlorinc]

Hi Maco,

I see you have a single-end BAM file, but using paired-end mode for quantification.

Could you try removing the "-p" flag? It should work then.

Best wishes Lorinc

On Mon, May 30, 2022, 8:05 AM marco91sol @.***> wrote:

Hi,

I am using TPMCalculator using the following command:

TPMCalculator -g file.gtf -d ./ -a -p -e

In the current directory, I have sorted bam files deriving from HISAT2 alignment tool of RNA reads against the genome.

Here, you will find the head of samtools view on bam file. [image: image] https://user-images.githubusercontent.com/42739946/170988264-b268bd7b-8eb4-4eef-8141-11d3cc4bbdfa.png

Here, follows part of the log file:

Reading GTF file ... Done in 12.0684 seconds Parsing sample: trimmed_hisat_out.bam.sorted 0 reads processed in 23.4511 seconds Printing results Total time: 42.3443 seconds

Thanks for the support!

Br, Marco

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[marco91sol]

It leaves me the following output: Chromosome with name: NW_001823611.1 does not exist

Is it normal?

[pongorlorinc]

Hmmm, it means that there is a mismatch of chromosomes between the gtf and bam file.

Did you use the same gtf during alignment?

On Mon, May 30, 2022, 9:19 AM marco91sol @.***> wrote:

It leaves me the following output: Chromosome with name: NW_001823611.1 does not exist

Is it normal?

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[marco91sol]

At the end, it worked even if there was the sentence "Chromosome with name: ... does not exist"

But, in any case, I have some issues with other bam files.

I'm not figuring out what's the issue. I have tried with some bam files describing paired reads/single end reads mapping genome. Could be an issue related to the reads name?

Thanks, Marco

[pongorlorinc]

What error do you get with this other bam file?

On Tue, May 31, 2022, 4:52 AM marco91sol @.***> wrote:

At the end, it worked even if there was the sentence "Chromosome with name: ... does not exist"

But, in any case, I have some issues with other bam files.

[image: image] https://user-images.githubusercontent.com/42739946/171132948-5e1c5313-053a-4ef7-8f0b-3bd54a89a464.png

I'm not figuring out what's the issue. I have tried with some bam files describing paired reads/single end reads mapping genome. Could be an issue related to the reads name?

Thanks, Marco

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[marco91sol]

No error. It only gives me this as output: """ Reading GTF file ... Done in 6.53447 seconds Parsing BAM files 0 reads processed in 9.5e-05 seconds Printing results Total time: 6.53495 seconds"""

[pongorlorinc]

Seems like the bam file is not sorted. That may cause an issue. Could you sort the bam file and retry quantification?

On Tue, May 31, 2022, 8:07 AM marco91sol @.***> wrote:

No error. It only gives me this as output: """ Reading GTF file ... Done in 6.53447 seconds Parsing BAM files 0 reads processed in 9.5e-05 seconds Printing results Total time: 6.53495 seconds"""

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[marco91sol]

At the moment, It's working... There were some issues with the samtools sort command.

However, It provides the usual mistake " Chromosome with name: NW_020844013.1 does not exist". Neverthless it worked before even giving that mistake.

Thanks for the support!

Best regards, Marco

[pongorlorinc]

Glad to hear it!

On Tue, May 31, 2022 at 10:42 AM marco91sol @.***> wrote:

At the moment, It's working... There were some issues with the samtools sort command.

However, It provides the usual mistake " Chromosome with name: NW_020844013.1 does not exist". Neverthless it worked before even giving that mistake.

Thanks for the support!

Best regards, Marco

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